Thymidlyate synthetase is the target of certain clinically useful cancer chemotherapeutic agents. The objective of this proposal is the elucidation of mechanistic features of the thymidylate synthetase reaction. Any insight into the mechanism of an enzymic reaction requires a thorough knowledge of the mechanistic features of analogous model (non-enzymic) reactions. Therefore, the first phase of our studies involved the development of congruent chemical models of the enzymic reaction, and a detailed investigation of the mechanisms of the chemical conversions of interest. The current phase involves studies with the purified enzyme in which we shall attempt to verify salient features of our model studies and uncover aspects not observable in the chemical counterparts. We have also initiated studies using in vivo (tissue culture) systems. We shall continue our investigation of the interaction of 5-fluoro-2'-deoxyuridylate and the cofactor with thymidylate synthetase. This inhibitor forms a covalent bond with the enzyme and cofactor, and acts "quasi substrate" which proceeds through a portion of the catalytic sequence before the blockade. We shall also continue studies of the interaction of other inhibitors which behave in a similar manner, such as 5-trifluoromethyl-2'-deoxyuridylate. We are continuing studies of secondary isotope effects in the enzyme catalyzed reaction in an attempt to further elucidate the mechanism of the enzyme and are investigating false substrates such as 5-bromo- and 5-iodo-2'-deoxyuridylate. We shall also perform binding studies of substrates and products and analogs with the enzyme. We are extending our work to in vivo systems to ascertain whether the outcome of our biological studies are useful in optimizing inhibiton of thymidylate synthetase. We shall also investigate a number of enzymes we feel share common mechanistic features with thymidylate synthetase.